cGMP and cAMP-dependent protein kinases (PKG and PKA) are closely related homologs, and the cyclic nucleotide specificity of each kinase is crucial for keeping the two signaling pathways segregated, but the molecular mechanism of cyclic nucleotide selectivity is unknown. Here we report that the PKG Iβ C-terminal cyclic nucleotide binding domain (CNB-B) is highly selective for cGMP binding, and have solved crystal structures of CNB-B with and without bound cGMP. These structures, combined with a comprehensive mutagenic analysis, allowed us to identify Leu296 and Arg297 as key residues which mediate cGMP selectivity. In addition, by comparing the cGMP bound and unbound structures, we observed large conformational changes in the C-terminal helices in response to cGMP binding, which were stabilized by recruitment of Tyr351 as a “capping residue” for cGMP. The observed rearrangements of the C-terminal helices provide a mechanical insight into release of the catalytic domain and kinase activation.

The Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) is a key regulator across the malaria parasite life cycle. Little is known about PfPKG’s activation mechanism. Here we report that the carboxyl cyclic nucleotide binding domain functions as a “gatekeeper” for activation by providing the highest cGMP affinity and selectivity. To understand the mechanism, we have solved its crystal structures with and without cGMP at 2.0 and 1.9 Å, respectively. These structures revealed a PfPKG-specific capping triad that forms upon cGMP binding, and disrupting the triad reduces kinase activity by 90%. Furthermore, mutating these residues in the parasite prevents blood stage merozoite egress, confirming the essential nature of the triad in the parasite. We propose a mechanism of activation where cGMP binding allosterically triggers the conformational change at the αC-helix, which bridges the regulatory and catalytic domains, causing the capping triad to form and stabilize the active conformation.

SUMMARY Cyclic GMP-dependent protein kinase (PKG) is a key regulator of smooth muscle and vascular tone and represents an important drug target for treating hypertensive diseases and erectile dysfunction. Despite its importance, its activation mechanism is not fully understood. To understand the activation mechanism, we determined a 2.5 Å crystal structure of the PKG I regulatory (R)-domain bound with cGMP, which represents the activated state. Though we used a monomeric domain for crystallization, the structure reveals that two R-domains form a symmetric dimer where the cGMP bound at high affinity pockets provide critical dimeric contacts. Small angle X-ray scattering and mutagenesis support this dimer model suggesting that the dimer interface modulates kinase activation. Finally, structural comparison with the homologous cAMP-dependent protein kinase reveals that PKG is drastically different from PKA in its active conformation, suggesting a novel activation mechanism for PKG.

Background: Protein kinase G (PKG) is selectively activated by cGMP, but the mechanism of cGMP-versus-cAMP selectivity is not fully understood. Results: cAMP-bound PKG exists in a dynamic exchange between three states, inactive, active, and a partially autoinhibited state, which results in partial agonism. Conclusion: Partial agonism contributes to cGMP-versus-cAMP selectivity. Significance: The cGMP-versus-cAMP selectivity controls the cross-talk between cGMP-and cAMP-dependent signaling pathways.

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